Field of Invention
This invention relates to a novel method for assessing the flavor quality of a juice and/or cider as well as the safety of a juice and/or cider by determining the quantity of microorganisms' DNA present in the juice and/or cider. This invention relates to a novel method of isolating DNA from a juice and/or cider.
Brief Description of the Prior Art
Huanglongbing (HLB) disease in Florida is widespread and associated with Candidatus Liberibacter asiaticus (CLas), a phloem limited bacterium. This disease can kill a tree in 5-10 years, and orange juice processed from HLB affected fruit is often associated with bitter taste and/or off-flavor (Baldwin, et al., J. Agr. Food Chem. 58:1247-1262 (2010); Plotto, et al., J. Food Sci. 75:S220-S230 (2010)). CLas population has been shown to correlate with HLB symptoms in that leaves with serious symptoms had higher CLas population (Gottwald, et al., Crop Protect. 36:73-82 (2012); Stover and McCollum, HortScience 46:1344-1348 (2011); Trivedi, et al., European J. Plant Pathol. 124:553-563 (2009)). Candidatus Liberibacter africanus (CLaf) and Candidatus Liberibacter americanus (CLam) also cause HLB disease in citrus trees in Africa and Brazil, respectively.
Quantitative polymerase chain reaction (qPCR) is an excellent diagnostic assay tool for determining if a plant is infected with a pathogen. Prior to conducting qPCR, one must first isolate and purify DNA. While many different methods of isolating and purifying DNA exist, in general, the most commonly used methods fall within one of two categories. One category uses SDS to lyse the cells, then uses a high concentration of KoAC to precipitate SDS and protein and thus is referred to as the SDS-KoAC method (Dellaporta, et al., Plant Molecular Biology Reporter, 1(4):19-21(1983)). The second category uses CTAB in the extraction procedure and thus is referred to as the CTAB method (Allen, et al., Nat. Protoc., 1(5):2320-2325 (2006)). Many additional methods exist including, but not limited to, silica-based isolation kits, magnetic particle separation technology, use of resins, etc. One of the difficulties in isolating DNA from plant material and pathogen cells in the presence of rigid polysaccharide cell wall and capsules which physically inhibit DNA liberation (Gonzalez-Mendoza, et al., Gen. Mol. Res. 9:162-166 (2010); Noor Adila, et al., Mal. J. Microbiol. 3:7-13 (2007); Varma, et al., Biotechnol. J. 2:386-392 (2007)). The most widely used method for tissue disruption for DNA extraction from plant tissue has been by grinding tissue with mortar and pestle under liquid nitrogen: the finer the grind, the greater the amount of DNA extracted (Rogstad, et al., Plant Mol. Biol. Rep. 19:353-359 (2001)). When the concentration of target DNA is low and the concentration of interfering compounds (e.g., plant cell walls, pectin, other sugars, proteins, secondary metabolites) is high, then additional lysis steps (e.g., mechanical disruption, sonication, enzymatic digestion) and/or additional purification steps (e.g., elution column) may be required (Al-Samarrai and Schmid, Lett. Appl. Microbiol. 30:53-56 (2000); Alaey, et al., Intl. J. Agr. Biol. 7:882-884 (2005); and Gonzalez-Mendoza, et al. (2010)). However, when one uses orange tree leaves or more precisely, midribs of orange tree leaves, which are rich in phloem vessels that harbor the CLas bacteria, one is able to isolate and purify bacterial DNA without using a sonicator, digestive enzymes or an elution column because the concentration of CLas DNA is high in phloem vessels compared to the concentration of the above described interfering compounds.
In contrast to midribs of leaves, juice vesicles contain a much lower amount of CLas cells (0.25% that of citrus leaf tissue) (Li, et al., Plant Dis. 92:854-861 (2008); Liao and Burns, J. Expt. Bot. 63:3307-3319 (2012)) and more interfering compounds (e.g., pectin, other sugars and secondary metabolites) (Baldwin et al., (2010)). Orange juice has extremely high pectin content compared to leaves and even other juices, measured as galacturonic acid (0.037-1.433 mg/g), depending on variety and harvest time (Baldwin, et al. (2010)). The pectin and DNA often co-purify when using prior art methods of DNA isolation (Scott and Playford, BioTechniques 20:974-978 (1996)). In addition, the other sugars in orange juice (e.g., sucrose, glucose and fructose) interfere with DNA extraction and isolation when using prior art isolation methods (Baldwin, et al. (2010)). A number of DNA extraction methods have been developed to avoid the co-precipitation of pectin/polysaccharides and DNA, including the use of high NaCl concentration (Varma, et al. (2007)) in conjunction with modified cetyl trimethyl ammonium bromide (CTAB) (Shepard and McLay, J. Plant Res. 124:311-314 (2011)), phenol, ethylene glycol monoethyl ester and pectinase (Okada, et al., Amer. J. Bot. 84:1236:1246 (1997); Rether, et al., Plant Mol. Biol. Rep. 11:333-337 (1993); and Rogstad, et al. (2001)). Unfortunately, although the CTAB plus high NaCL concentration method works well for isolating and purifying CLas DNA from citrus leaf tissue which has high CLas DNA concentration, this method does not work well with citrus juice which has low CLas DNA concentration and a high concentration of interfering polysaccharides, especially pectin. Another method uses pectinase to digest the pectin (Bai, et al., in press), however, only low yields of DNA are obtained (see Table 1) perhaps because commercial pectinase preparations contain low levels of DNase. Thus, it is difficult to obtain high quantity and quality DNA (both microorganism's DNA and plant DNA) from citrus juice. In fact, prior attempts to isolate sufficient quantity of DNA at a sufficient purity level from orange juice using Qiagen's DNeasy Plant Mini Kit, Qiagen's mericon Food Kit, Qiagen's QIAamp DNA Blood Mini Kit, or Promega's Wizard® Genomic DNA purification kit were not successful (see infra).
As mentioned above, orange juice is rich in secondary metabolites, including alkaloids, limonoids, and flavonoids, which are not present in high concentrations in leaf tissue relative to CLas DNA (Baldwin, et al. (2010); Justesen, et al., J. chromatogr. A 799:101-110 (1998)). These secondary metabolites can inhibit PCR reaction (Deng and Cliver, Intl. J. Food Microbiol. 54:155-162 (2000); Kim and Cho, Food Control 21:1419-1423 (2000); Ogunjimi and Choudary, FEMS Immunol. Med. Microbiol. 23:213-220 (1999); Tsai and Olson, Appl. Environ. Microbiol. 58:2292-2295 (1992); Wilson, I .G., Appl. Environ. Microbiol. 63:3741-3751 (1997)). Appropriate ion exchange columns or chelating agents can be used to remove these contaminants (Saunders, G. C., DNA extraction, p29-46. In: G. C. Saunders and H. C. Parkers (eds.) Analytical molecular biology: Quality and validation. Royal Soc. Chem. Cambridge). Kim and Cho (2010) successfully removed PCR inhibitors from apple, grape, and watermelon juices using Chelex treatment and Sephadex column filtration. However, Kim and Cho were unsuccessful using this method to isolate DNA free from secondary metabolites from orange juice. In contrast, Li, et al. (J. Microbiol. Meth. 66:104-115 (2006)) showed that TaqMan® and other commercial “real-time” PCR assays for CLas DNA were not inhibited when the samples were extracted from citrus leaf tissue with the standard cetyl trimethyl ammonium bromide (CTAB) method or the DNeasy® plant kit (Qiagen, Gaithersburg, Md.), indicating that these qPCR assays with a small amplicon (about 70 bp) perhaps are less vulnerable to inhibitors of the amplification reaction in comparison with the conventional PCR assays with a large amplicon (about 1200 bp) (Mackay, et al., Nucl. Acid. Res. 30:1292-1305 (2002)). The TaqMan® assay was also used with orange juice and successfully amplified CLas DNA. However, as mentioned previously, these DNA extraction methods yield too low a concentration of DNA to be of practical use. Furthermore, standard deviations of the cycle threshold (Ct) value in qPCT increases as the quantity of target DNA decreases, indicating a higher risk of error at low target DNA concentrations (Liu, et al., J. Microbiol. Meth. 65:21-31 (2006)).
In 2013, a new method for detecting CLas DNA in orange juice was disclosed (Bai, et al., Proc. Fla. State Hort. Soc. 125:233-238 (2013)), however this method is too complicated for commercial use by citrus processors, uses too much orange juice during the assay, and is not consistently accurate because of the small amount of DNA produced results in an increase of the standard deviation of Ct value. As such, the need still exists for a simple, reliable assay to detect CLas in citrus juice by assaying for CLas DNA in the juice. Furthermore, this method enables one to isolate and purify the DNA of other microorganisms in orange juice and other juices.
Currently, orange juice quality is determined by a U.S. Department of Agriculture grades as determined by an inspector who tastes and grades the juice and by measurement of soluble solids, titratable acidity and sometimes the bitter limonoid, limonin by the juice processors. There is a need for a more quantitative assay to determine whether the quality of a juice has been adversely impacted by a microorganism, and if so, how much has the flavor changed. This assay can also be used to determine if a juice is fit for consumption.